Throughout this application various publications are referred to in short form. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Clostridium difficile, an anaerobic spore-forming bacterium, was found in the 1970s to be the cause of pseudomembranous colitis, and the major agent of antibiotic-associated diarrhea, causing about 20% of cases (Dubberke et al. 2009, Kelly, 2008). The diarrheal disease is due to expression of major toxins A and B by “toxigenic” C. difficile strains; other strains of this bacterium do not contain the genes for these toxins and are entirely non-pathogenic (Kelly, 2008). Infectious spores of C. difficile can survive in the environment for months, resist drying and many common disinfectants, and thus represent a major reservoir of this organism within hospitals (Dubberke et al. 2009). With the arrival of a new “hypervirulent” strain in the past decade, the incidence of C. difficile disease and its severity have increased dramatically (Kelly 2008, Dubberke et al. 2009). C. difficile is now the leading hospital-acquired infection in some regions of North America, affecting over 1 out of 100 hospitalized adults (APIC, 2008, Jarvis et al., 2009). There were an estimated 300,000 infections in acute care hospitals in the US annually in 2005 (Dubberke et al. 2009) and an equal number in long-term care facilities (Ohio DOH, 2007). While in the past these infections were merely a nuisance, the new C. difficile now has an attributable mortality of ˜7%, and contributes to death in another 7.5% of patients (Kelly 2008). A further problem is that current treatments are plagued by a 20-50% recurrence rate, with many patients continuing to relapse with this condition for years (Kelly 2008, Dubberke et al. 2009). The overall costs of C. difficile disease are estimated to be as high as $3.2 billion annually (Dubberke et al. 2009, Cohen et al. 2010).
Diagnosis of C. difficile: Clostridium difficile disease was initially identified by anaerobically growing the organism on selective culture media (George et al. 1979; Buggy et al. 1983)), which required 2 days and failed to distinguish between toxigenic and nontoxigenic strains. The presence of toxins needed to be confirmed from culture; this involved a 48 hr cytotoxicity assay using tissue cultured epithelial cells; specificity of cytotoxicity was demonstrated by its neutralization by Clostridia-specific anti-toxin. Alternatively, cytotoxicity could be detected directly in preparations of stool samples (George et al. 1979). However, due to the labor-intensive and technically complex nature of this assay, it was quickly replaced in the 1990s by Enzyme-linked Immunosorbent Assays (EIAs or ELISAs) directed at the toxin A, or later toxin A & B antigens (Planche et al. 2008, Cohen et al. 2010). While in experimental settings, these assays had sensitivities reported to be over 90%, recent evidence shows that in day-to-day practice, their sensitivity was reduced to below 50% (Ticehurst et al. 2006, Reller et al. 2007, Fenner et al. 2008, Sloan et al. 2008, Stamper et al. 2009). Why this discrepancy occurs is not clear, though it may relate to the lability of C. difficile toxins in clinical samples, or possibly changes in antibody affinity for their epitopes. Failure to detect C. difficile, in the current epidemic is no longer acceptable, so alternative strategies for diagnosis are needed.
One approach involves identifying samples that contain C. difficile by using an EIA for an abundant, stable antigen, the glutatamate dehydrogenase (gdh) protein (Ticehurst et al. 2006, Fenner et al. 2008, Gilligan 2008). This test is rapid, much more sensitive than toxin A/B EIA, but fails to discriminate toxigenic from non-toxigenic strains. Thus, while its useful in eliminating patients without C. difficile (due to a high negative predictive value) (Reller et al. 2007, Kvach et al. 2010), its positive predictive value is low—about ⅓ of those positive by gdh EIA do not have toxigenic strains. Thus, those patients found to have a positive gdh EIA need to be assessed by alternate means, which could be an insensitive toxin EIA or the traditional 48 hr cytotoxicity tissue culture assay
Another approach for detecting toxigenic C. difficile involves amplification of the toxin B gene by polymerase chain reaction (PCR). Commercial reagents for PCR directly from stool have been FDA-approved from several manufacturers in the past year, and these assays are rapid, sensitive and specific (Stamper et al. 2009, Kvach et al. 2010). However, they are costly (up to 10 times the cost of the EIA test), and require some technical expertise.
Finally, toxigenic culture remains the gold standard by which most diagnostic assays are judged, as acknowledged by recent national guidelines (Cohen, 2010). It has not been widely adapted due to i) the delay in getting results (2 to 7 days) and ii) the technical expertise and equipment required (often including an anaerobic chamber). Nonetheless, various modifications of toxigenic culture have consistently demonstrated the highest accuracy in detecting C. difficile infection. While most reported studies have focused on solid agar cultures, which allow colony morphology to guide identification, there have been several reports of liquid culture used for diagnosis. In the 1980s, chopped meat broth was utilized to maintain anaerobiosis, and accurate detection of C. difficile was reported within 24-48 hrs using gas liquid chromatography to detect the volatile amines characteristic of this organism (Buchanan 1984, Johnson et al. 1989). 225 of 226 strains were detected by this method; however, these included nontoxigenic strains (Johnson et al. 1989).
A modification of the basic CCFA agar was used to create a broth, but its performance was no better than the agar (Clabots et al. 1991). Recently, a re-examination of this broth, with incorporation of the bile salt taurocholate to promote germination of spores (Buggy et al. 1983), showed its utility for clinical and environmental isolates (Arroyo et al. 2005, Neranzdic et al. 2009). A commercial form of this broth exists (Anaerobe Systems AS-8216, Morgan Hill, Calif.), but it requires an anaerobic environment for culture, limiting its general applicability. Again, most forms of culture require confirmation of cytotoxin production, which has been usually done using the 48 hr cytotoxicity assay.
The present invention addresses the need for improved methods for diagnosis of toxigenic C. difficile. 